Dataset Information

Integrative proteomics and phosphoproteomics profiling and systems biology analysis reveal dynamic signaling networks and mTORC1-dependent mitochondrial function in T cell activation

ABSTRACT: The underlying mechanisms by which naïve T cells exit from quiescence after antigen stimulation remain elusive. Using multiplex isobaric labeling proteomics technology, we report unbiased, temporal profiling of whole proteome (>8,000 proteins) and phosphoproteome (>13,000 phosphopeptides) during the activation in the wild type and Rptor-/- T cells. TCR stimulation results in dynamic reprogramming of the proteome and phosphoproteome, with predominant upregulation of molecular machineries in protein translation and mitochondrial activation. Loss of mTORC1 disrupts TCR-induced mitochondrial functions including mitoribosome biogenesis, one-carbon metabolism and oxidative phosphorylation.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): T Cell

SUBMITTER: xusheng wang

LAB HEAD: Junmin Peng

PROVIDER: PXD004367 | Pride | 2017-03-15

REPOSITORIES: Pride

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Publications

Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

Tan Haiyan H Yang Kai K Li Yuxin Y Shaw Timothy I TI Wang Yanyan Y Blanco Daniel Bastardo DB Wang Xusheng X Cho Ji-Hoon JH Wang Hong H Rankin Sherri S Guy Cliff C Peng Junmin J Chi Hongbo H

Immunity 20170309 3

The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early d ...[more]

PMID: 28285833

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Project description:To investigate the mechanism by which Cul5 regulates CD8+ T cell cytokine-dependent differentiation and TCR-dependent activation, we performed quantitative mass spectrometry (MS) by data-independent acquisition (DIA)-MS of total proteins in the Cul5 KO and NC primary CD8+ T cells in the following conditions: 1) Cytokine dependent expansion and differentiation (T0); 2) 8 hours cytokine withdraw prior to TCR stimulation (T8); and 3) 16 hours TCR stimulation post 8-hour cytokine withdraw (T16). Principal component analysis (PCA) and correlation analysis revealed that replicates in each condition clustered together while different conditions separated from each other, suggesting significant proteomic changes among different conditions of the same T cells as well as between Cul5 KO and NC T cells in each condition. Together with similar total protein quantities among all detected samples, DIA-MS analyses were of high quality. Additionally, the strong reductions of Cul5 abundances in the Cul5 KO cells from all three conditions compared to the NC cells confirms high KO efficiency. Of note, the proteomic analysis showed that the Cul5 protein level was significantly increased upon TCR stimulation post cytokine starvation in the NC cells, suggesting a potential negative feedback regulatory role of Cul5 in CD8+ T cell activation. Consistent with this idea, we observed more markedly upregulation of Cul5 expression upon of TCR stimulation of na•ve primary CD8+ cells. To identify Cul5 interacting proteins in CD8+ T cells, we overexpressed Cul5 with a C terminal HA-tag (Cul5-HA) in mouse primary CD8+ T cells by retroviral transduction. The cells were subjected to TCR stimulation for 12 hr, and Cul5-HA was immunoprecipitated by anti-HA, followed by DIA-MS analysis (co-IP-MS). Compared to the negative control samples (cells transduced with the empty vector), 65 proteins were enriched (p value <0.05 and fold change >1.5) in the anti-HA IP samples. Altogether, we report that Cul5 KO alters CD8+ T cell proteome and the Cul5 interactome.

Project description:Though T cell expansion and effector differentiation are triggered and, perhaps, maintained by antigen, the proliferative behaviors of CD4+ and CD8+ T cells responding to timed antigen presentation have rarely been compared side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal T cells were analyzed following transient and continuous TCR signals. We found CD4+ T cell proliferation to be dependent on prolonged antigen presence, whereas CD8+ T cells were able to divide and differentiate into effector cells in the absence of it. We excluded CD4+ T cell proliferation to be abrogated by coinhibitory signals or the lack of inflammatory stimuli and found that autonomous proliferation of CD8* T cells was independent of any MHC class I signals. Gene expression analyses illustrated differences in global gene transcription between the two subsets following stimulation periods of different lengths. These T cell data reflect the MHC class difference in that class II but not class I molecules were stabilized on activated DCs in vivo, suggesting a coevolution of MHC molecules and their respective T cell subsets. Samples 1-12: Analysis on day 2. Purified CD4+ AND-TCR transgenic cells andÂ CD8+ OT1-TCR transgenic cells were separately stimulated with anti-CD3 and anti-CD28 antibodies. 48 hours later, the cells were sorted again to a purity of >99 %. Extracted total RNA was amplified twice and hybridized on Affymetrix Mouse 430A2 microarrays. First, we analysed the changes of the CD4+ and CD8+ T cells after stimulation. Second, we compared the differences of the changes between the two cell types after stimulation. For each of the four groups (CD4+ and CD8+, stimulated and unstimulated), we analysed three independent biological replicates. Samples 13-28: Analysis on day 5. AND and OT1 TCR-transgenic T cells were prepared as described before, but transferred into mice that do not or do present their respective antigens. 72 hours later, the cells were FACS-sorted twice to >99 % purity, directly into Trizol. For each of the six groups (CD4+ and CD8+, unstimulated, transient (2 days) and continuous (5 days) stimulation), three independent biological replicates were analyzed, except for CD4+ unstimulated and CD4+ transient, with two replicates each.

Dataset Information

Integrative proteomics and phosphoproteomics profiling and systems biology analysis reveal dynamic signaling networks and mTORC1-dependent mitochondrial function in T cell activation

Publications

Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

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